Dna sequence, and recombinant preparation of the grass pollen allergen lol p 4

ABSTRACT

The present invention relates to the provision of a DNA sequence of the major grass pollen allergen Lol p 4. The invention also encompasses fragments, new combinations of partial sequences and point mutants having a hypoallergenic action. The recombinant DNA molecules and the derived polypeptides, fragments, new combinations of partial sequences and variants can be utilised for the therapy of pollen-allergic diseases. The proteins prepared by recombinant methods can be employed for in vitro and in vivo diagnosis of pollen allergies.

BACKGROUND OF THE INVENTION

The present invention relates to the provision of a DNA sequence of themajor grass pollen allergen Lol p 4. The invention also encompassesfragments, new combinations of partial sequences and point mutantshaving a hypoallergenic action. The recombinant DNA molecules and thederived polypeptides, fragments, new combinations of partial sequencesand variants can be utilised for the therapy of pollen-allergicdiseases. The proteins prepared by recombinant methods can be employedfor in vitro and in vivo diagnosis of pollen allergies.

Type 1 allergies are of importance worldwide. Up to 20% of thepopulation in industrialised countries suffer from complaints such asallergic rhinitis, conjunctivitis or bronchial asthma. These allergiesare caused by allergens present in the air (aeroallergens) which arereleased by sources of various origin, such as plant pollen, mites, catsor dogs. Up to 40% of these type 1 allergy sufferers in turn exhibitspecific IgE reactivity with grass pollen allergens (Freidhoff et al.,1986, J. Allergy Clin. Immunol. 78, 1190-2001).

The substances which trigger type 1 allergy are proteins, glycoproteinsor polypeptides. After uptake via the mucous membranes, these allergensreact with the IgE molecules bonded to the surface of mast cells insensitised individuals. If two IgE molecules are crosslinked to oneanother by an allergen, this results in the release of mediators (forexample histamine, prostaglandins) and cytokines by the effector celland thus in the corresponding clinical symptoms.

A distinction is made between major and minor allergens, depending onthe relative frequency with which the individual allergen moleculesreact with the IgE antibodies of allergy sufferers.

For perennial ryegrass (Lolium perenne), Lol p 1 has been identified asa major allergen (Freidhoff et al., 1986, J. Allergy Clin. 78:1190-1201)and its primary structure has been elucidated (Perez et al., 1990, J.Biol. Chem. 265:16210-16215). A further major allergen is Lol p 2(Freidhoff et al., 1986, J. Allergy Clin. 78:1190-1201), the primarystructure of which was described in 1993 (Ansari et al., 1989, J. Biol.Chem.: 264:11181-11185). A further major allergen of perennial ryegrassis Lol p 5 (Mattiesen and Löwenstein 1991, Clin. Exp. Allergy 21:297-307). The primary structure of Lol p 5 is also known (Ong et al.,1993, Gene 134:235-240). Perennial ryegrass furthermore contains themajor allergens from groups 4 (Fahlbusch et al. 1998, Clin. Exp. Allergy28: 799-807) and 13 (Petersen et al., 2001, J. Allergy Clin. 1 mm.107:856-862).

Lol p 4 is a typical basic glycoprotein (Jaggi et al, 1989, Int. Arch.Allergy Appl. Immunol. 89:342-348, Jaggi et al., 1989, J. Allergy Clin.Immunol. 83:845-852) and is comparable with the well-studied PhI p 4,Cyn d 4 and Dac g 4 in terms of cross-reactivity with specific IgEantibodies (Haavik et al., 1985, Int. Arch. Allergy Appl. Immunol.78:260-268; Su et al., 1991, Clin. Exp. Allergy 21:449-455;Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98:1065-1072;14-17).

These homologous molecules from the Poaceae form allergen group 4, themolecules of which have high immunological cross-reactivity with oneanother both with monoclonal murine antibodies and also with human IgEantibodies (Fahlbusch et al., 1993 Clin. Exp. Allergy 23:51-60;Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98:1065-1072; Suet al., 1996, J. Allergy Clin. Immunol. 97:210; Fahlbusch et al., 1998,Clin. Exp. Allergy 28:799-807; Gavrovic-Jankulovic et al., 2000, Invest.Allergol. Clin. Immunol. 10 (6):361-367; Stumvoll et al. 2002, Biol.Chem. 383:1383-1396; Grote et al., 2002, Biol. Chem. 383:1441-1445;Andersson and Lidholm, 2003, Int. Arch. Allergy Immunol. 130:87-107;Mari, 2003, Clin. Exp. Allergy, 33 (1):43-51).

In contrast to the above-mentioned major allergens Lol p 1, Lol p 2, Lolp 5 from Lolium perenne, the primary structure of Lol p 4 has not yetbeen elucidated.

From the group 4 allergen from Dactylus glomerate, it has hitherto onlybeen possible for peptides to be obtained by enzymatic degradation andsequenced:

DIYNYMEPYVSK (SEQ ID NO 7), VDPTDYFGNEQ (SEQ ID NO 8), ARTAWVDSGAQLGELSY(SEQ ID NO 9)

and GVLFNIQYVNYWFAP (SEQ ID NO 10, Leduc-Brodard et al., 1996, J.Allergy Clin. Immunol. 98: 1065-1072).Peptides have also been obtained from the group 4 allergen ofsub-tropical Bermuda grass (Cynodon dactylon) by proteolysis andsequenced:

KTVKPLYIITP (SEQ ID NO 11), KQVERDFLTSLTKDIPQLYLKS (SEQ ID NO 12),TVKPLYIITPITAAMI (SEQ ID NO 13), LRKYGTAADNVIDAKVVDAQGRLL (SEQ ID NO14), KWQTVAPALPDPNM (SEQ ID NO 15), VTWIESVPYIPMGDK (SEQ ID NO 16),GTVRDLLXRTSNIKAFGKY (SEQ ID NO 17), TSNIKAFGKYKSDYVLEPIPKKS (SEQ ID NO18), YRDLDLGVNQWG (SEQ ID NO 19), SATPPTHRSGVLFNI (SEQ ID NO 20),

and AAAALPTQVTRDIYAFMTPYVSKNPRQAYVNYRDLD (SEQ ID NO 21, Liaw et al.,2001, Biochem. Biophys. Research Communication 280: 738-743).

For Lolium perenne, peptide fragments having the following sequenceshave been described for the basic group 4 allergen: FLEPVLGLIFPAGV (SEQID NO 22) and GLIEFPAGV (SEQ ID NO 23, Jaggi et al., 1989, Int. Arch.Allergy Appl. Immunol. 89: 342-348).

However, these peptide sequences which have been described for Loliumperenne and other group 4 allergens have hitherto not resulted in theelucidation of the primary structure of the Lol p 4 allergen.

As the first sequence of a group 4 allergen, the still unpublishedsequence of PhI p 4 from Phleum pratense has been elucidated by theinventors of the present patent application and described inInternational Application WO 04/000881.

The object on which the present invention is based therefore consistedin the provision of a DNA sequence of the Lol p 4 gene encoding anallergen having the immunological properties of Lol p 4, and acorresponding recombinant DNA, on the basis of which the allergen can beexpressed as protein and made available, as such or in modified form,for pharmacologically significant exploitation. The sequence of Phl p 4was the starting point for the present, invention.

List of Sequences According to the Invention

-   -   DNA sequence from the Lol p 4 gene (SEQ ID NO 1).    -   Protein sequence derived from the DNA sequence in accordance        with SEQ ID NO 1 (SEQ ID NO 2).    -   DNA sequence (SEQ ID NO 3), composed of nucleotides 1-200 of Phl        p 4 (in accordance with SEQ ID NO 5), 201-1472 of Lol p 4 (in        accordance with SEQ ID NO 1) and 1473-1503 of Phl p 4 (in        accordance with SEQ ID NO 5).    -   Protein sequence (SEQ ID NO 4), composed of amino acids 1-67 of        Phl p 4 (in accordance with SEQ ID NO 6), 68-490 of Lol p 4 (in        accordance with SEQ ID NO 2) and 491-500 of Phl p 4 (in        accordance with SEQ ID NO 6) having the properties, in        particular immunological properties, of Lol p 4, encoded by the        DNA sequence in accordance with SEQ ID NO 3.    -   DNA sequence of Phl p 4 (SEQ ID NO 5), in accordance with SEQ ID        NO 5 from WO 04/000881.    -   Protein sequence of Phl p 4 (SEQ ID NO 6), in accordance with        SEQ ID NO 6 from WO 04/000881.

DESCRIPTION OF THE INVENTION

The present invention provides for the first time a DNA sequence of themajor grass pollen allergen Lol p 4 (SEQ ID NO 1) which encodes anallergen having the immunological properties of Lol p 4.

The present invention therefore relates to a DNA molecule encoding anallergen having the properties of Lol p 4, corresponding to a nucleotidesequence in accordance with SEQ ID NO 1.

The invention furthermore relates to a DNA molecule encoding an allergenhaving the properties of Lol p 4, corresponding to a nucleotide sequencein accordance with SEQ ID NO 3, composed of nucleotides 1-201 of Phl p 4(in accordance with SEQ ID NO 5), 202-1470 of Lol p 4 (SEQ ID NO 1) and1471-1500 of Phl p 4.

The invention furthermore relates to sequences homologous to the DNAsequences according to the invention and corresponding DNA molecules ofgroup 4 allergens from other Poaceae, such as, for example, Dactylisglomerata, Poa pratensis, Cynodon dactylon, Holcus lanatus, Secalecerale, Triticum aestivum and Hordeum vulgare, which, owing to thesequence homology that exists, hybridise with the DNA sequencesaccording to the invention under stringent conditions, or haveimmunological cross-reactivity with respect to Lol p 4.

The invention also encompasses fragments, new combinations of partialsequences and point mutants having a hypoallergenic action.

The invention therefore furthermore relates to corresponding partialsequences, a combination of partial sequences, or replacement,elimination or addition mutants which encode an immunomodulatory,T-cell-reactive fragment of a group 4 allergen from the Poaceae.

With knowledge of the DNA sequence of the naturally occurring allergens,it is now possible to prepare these allergens as recombinant proteinswhich can be used in the diagnosis and therapy of allergic diseases(Scheiner and Kraft, 1995, Allergy 50: 384-391).

A classical approach to effective therapeutic treatment of allergies isspecific immunotherapy or hyposensitisation (Fiebig, 1995, Allergo J. 4(6): 336-339, Bousquet et al., 1998, J. Allergy Clin. Immunol. 102 (4):558-562). In this method, the patient is injected subcutaneously withnatural allergen extracts in increasing doses. However, there is a riskin this method of allergic reactions or even anaphylactic shock. Inorder to minimise these risks, innovative preparations in the form ofallergoids are employed. These are chemically modified allergen extractswhich have significantly reduced IgE reactivity, but identical T-cellreactivity compared with the untreated extract (Fiebig, 1995, Allergo J.4 (7): 377-382).

Even more substantial therapy optimisation would be possible withallergens prepared by recombinant methods. Defined cocktails ofhigh-purity allergens prepared by recombinant methods, optionallymatched to the individual sensitisation patterns of the patients, couldreplace extracts from natural allergen sources since these, in additionto the various allergens, contain a relatively large number ofimmunogenic, but non-allergenic secondary proteins.

Realistic perspectives which may result in reliable hyposensitisationwith expression products are offered by specifically mutated recombinantallergens in which IgE epitopes are specifically deleted withoutimpairing the T-cell epitopes which are essential for therapy (Schrammet al., 1999, J. Immunol. 162: 2406-2414).

A further possibility for therapeutic influencing of the disturbed THcell equilibrium in allergy sufferers is immunotherapeutic DNAvaccination, which involves treatment with expressible DNA which encodesthe relevant allergens. Initial experimental evidence ofallergen-specific influencing of the immune response has been furnishedin rodents by injection of allergen-encoding DNA (Hsu et al., 1996,Nature Medicine 2 (5): 540-544).

The present invention therefore also relates to a DNA molecule describedabove or below as medicament and to a corresponding recombinantexpression vector as medicament.

The corresponding proteins prepared by recombinant methods can beemployed for therapy and for in vitro and in vivo diagnosis of pollenallergies.

For preparation of the recombinant allergen, the cloned nucleic acid isligated into an expression vector, and this construct is expressed in asuitable host organism. After biochemical purification, this recombinantallergen is available for detection of IgE antibodies by establishedmethods. The present invention therefore furthermore relates to arecombinant expression vector comprising a DNA molecule described aboveor below, functionally linked to an expression control sequence, and ahost organism transformed with said DNA molecule or said expressionvector.

The invention also relates to the use of at least one DNA moleculedescribed above or at least one expression vector described above forthe preparation of a medicament for the immunotherapeutic DNAvaccination of patients with allergies in the triggering of which group4 allergens from the Poaceae, in particular Lol p 4, are involved and/orfor the prevention of such allergies.

As already stated, the invention can be used as an essential componentin a recombinant allergen- or nucleic acid-containing preparation forspecific immunotherapy. A number of possibilities arise here. On the onehand, the protein with an unchanged primary structure may be aconstituent of the preparation. On the other hand, a hypoallergenic(allergoid) form can be used in accordance with the invention fortherapy in order to avoid undesired side effects by specific deletion ofIgE epitopes of the molecule as a whole or the production of individualfragments which encode T-cell epitopes. Finally, the nucleic acid perse, if ligated with a eukaryotic expression vector, gives a preparationwhich, when applied directly, modifies the allergic immune state in thetherapeutic sense.

The present invention furthermore relates to polypeptides encoded by oneor more of the DNA molecules described above, preferably in theirproperty as medicament. In particular, the polypeptides are a proteincorresponding to an amino acid sequence in accordance with SEQ ID NO 2or a protein which contains this amino acid sequence or a part of thissequence, having the properties, in particular immunological properties,of Lol p 4, and a protein corresponding to an amino acid sequence inaccordance with SEQ ID NO 4 having the properties, in particularimmunological properties, of Lol p 4.

Accordingly, the invention also relates to a process for the preparationof such polypeptides by cultivation of a host organism and isolation ofthe corresponding polypeptide from the culture.

The invention likewise relates to the use of at least one polypeptide orprotein described above for the preparation of a medicament for thediagnosis and/or treatment of allergies in the triggering of which group4 allergens from the Poaceae, in particular Lol p 4, are involved, andfor the prevention of such allergies.

When determining the protein and DNA sequence of Lol p 4, the followingprocedure was followed:

The Lol p 4 DNA sequence in accordance with SEQ ID NO 1 according to theinvention was amplified, cloned and sequenced by PCR with specificprimers (Table 1) derived from the Phl p 4 sequence in accordance withSEQ ID NO 5 as described in WO 04/000881. A total of 7 clones wereanalysed. Analysis of the clones gave a uniform sequence. Three Lol p 4DNA sequences were obtained by PCR with primers #87 and #83. The Lol p 4DNA sequence amplified with these primers encodes the correspondingamino acids 68-401, based on the numbering of mature Phl p 4 inaccordance with SEQ ID NO 6. Two further clones were obtained by PCRwith primers #87 and #189. The Lol p 4 DNA sequence amplified with theseprimers encodes the corresponding amino acids 68-490 (numberingcorresponding to Phl p 4 sequence). Two clones were obtained by PCR withprimers #87 and #131. The amplified Lol p 4 DNA sequence likewiseencodes the corresponding amino acids 68-490 (numbering corresponding toPhl p 4 sequence). Primers #131 and #189 correspond to the codons forthe final 10 amino acids of the Phl p 4 protein and span the stop codon.

The DNA sequence in accordance with SEQ ID NO 3 according to theinvention was obtained by methods known per se (PCR technique withoverlapping primers).

For the preparation of the recombinant allergens according to theinvention, the DNA sequences in accordance with SEQ ID NO 1 or 3 wereincorporated into expression vectors (for example pProEx, pSE 380). Forthe N-terminal amino acids known from the protein sequencing, E.coli-optimised codons were used.

After transformation in E. coli, expression and purification of therecombinant allergens according to the invention by various separationtechniques, the proteins obtained were subjected to a refolding process.

Both allergens can be employed for highly specific diagnosis of grasspollen allergies. This diagnosis can be carried out in vitro bydetection of specific antibodies (IgE, IgG1-4, IgA) and reaction withIgE-loaded effector cells (for example basophiles from blood) or in vivoby skin test reactions and provocation at the reaction organ.

The reaction of the allergens according to the invention withT-lymphocytes from grass pollen allergy sufferers can be detected byallergen-specific stimulation of the T-lymphocytes for proliferation andcytokine synthesis both with T-cells in freshly prepared bloodlymphocytes and also on established nLol p 4-reactive T-cell lines andclones.

The triplets encoding the cysteines were modified by site-specificmutagenesis in such a way that they encode other amino acids, preferablyserine. Both variants in which individual cysteines have been replacedand those in which various combinations of 2 cysteine residues or allcysteines have been modified were prepared. The expressed proteins ofthese cysteine point mutants have greatly reduced or zero reactivitywith IgE antibodies from allergy sufferers, but react with theT-lymphocytes from these patients.

The present invention therefore furthermore relates to a DNA moleculedescribed above or below in which one, a plurality of or all cysteineresidues of the corresponding polypeptide have been replaced by anotheramino acid by site-specific mutagenesis.

The immunomodulatory activity of hypoallergenic fragments whichcorrespond to polypeptides having T-cell epitopes and that of thehypoallergenic point mutants (for example cysteine replacements) can bedetected by their reaction with T-cells from grass pollen allergysufferers.

Such hypoallergenic fragments or point mutants of the cysteines can beemployed as preparations for hyposensitisation of allergy suffererssince they react with the T-cells with equal effectiveness, but resultin reduced IgE-mediated side effects owing to the reduced or entirelyabsent IgE reactivity.

If the nucleic acids encoding the hypoallergenic allergen variantsaccording to the invention or the unmodified DNA molecules according tothe invention are ligated with a human expression vector, theseconstructs can likewise be used as preparations for immunotherapy (DNAvaccination).

Finally, the present invention relates to pharmaceutical compositionscomprising at least one DNA molecule described above or at least oneexpression vector described above and optionally further activeingredients and/or adjuvants for the immunotherapeutic DNA vaccinationof patients with allergies in the triggering of which group 4 allergensfrom the Poaceae, in particular Lol p 4, are involved and/or for theprevention of such allergies. A further group of pharmaceuticalcompositions according to the invention comprises at least onepolypeptide described above instead of the DNA and is suitable for thediagnosis and/or treatment of said allergies.

Pharmaceutical compositions in the sense of the present inventioncomprise, as active ingredients, a polypeptide according to theinvention or an expression vector and/or respective pharmaceuticallyusable derivatives thereof, including mixtures thereof in all ratios.The active ingredients according to the invention can be brought into asuitable dosage form here together with at least one solid, liquidand/or semi-liquid excipient or adjuvant and optionally in combinationwith one or more further active ingredients.

Particularly suitable adjuvants are immunostimulatory DNA oroligonucleotides having CpG motives.

These compositions can be used as therapeutic agents or diagnosticagents in human or veterinary medicine. Suitable excipients are organicor inorganic substances which are suitable for parenteral administrationand do not adversely affect the action of the active ingredientaccording to the invention. Suitable for parenteral use are, inparticular, solutions, preferably oil-based or aqueous solutions,furthermore suspensions, emulsions or implants. The active ingredientaccording to the invention may also be lyophilised and the resultantlyophilisates used, for example, for the preparation of injectionpreparations. The compositions indicated may be sterilised and/orcomprise adjuvants, such as preservatives, stabilisers and/or wettingagents, emulsifiers, salts for modifying the osmotic pressure, buffersubstances and/or a plurality of further active ingredients.

Furthermore, sustained-release preparations can be obtained bycorresponding formulation of the active ingredient according to theinvention—for example by adsorption on aluminium hydroxide.

The invention thus also serves for improving in vitro diagnosis as partof allergen component-triggering identification of the patient-specificsensitisation spectrum. The invention likewise serves for thepreparation of significantly improved preparations for the specificimmunotherapy of grass pollen allergies.

TABLE 1 Primers used Primer SEQ ID number NO Sequence #83 24GGCTCCCGGGGCGAACCAGTAG #87 25 ACCAACGCCTCCCACATCCAGTC #131 26GATAAGCTTGAATTCTGATTAGTACTTTTTGATCAGCG GCGGGATGCTC #189 27GATAAGCTTCTCGAGTGATTAGTACTTTTTGATCAGCG GCGGGATGCTC

1. A DNA molecule encoding an allergen having the properties of Lol p 4,corresponding to a nucleotide sequence selected from one of thesequences in accordance with SEQ ID NO 1 and
 3. 2. A DNA molecule whichhybridises with a DNA molecule according to claim 1 under stringentconditions and originates from DNA sequences from Poaceae species.
 3. ADNA molecule, encoding a polypeptide, which cross-reacts immunologicallywith the major allergen Lol p 4 from Lolium perenne and originates fromDNA sequences from Poaceae species.
 4. A DNA molecule, corresponding toa partial sequence or a combination of partial sequences according toclaim 1, which encodes an immunomodulatory, T-cell-reactive fragment ofa group 4 allergen from the Poaceae.
 5. A DNA molecule, corresponding toa nucleotide sequence according to claim 1, encoding an immunomodulatoryT-cell-reactive fragment, characterised in that said nucleotide sequencehas been specifically modified by specific mutation of individualcodons, elimination or addition.
 6. A DNA molecule according to claim 5,characterised in that the said mutation results in the replacement ofone, a plurality of or all cysteines of the corresponding polypeptidewith another amino acid.
 7. A recombinant DNA expression vector or acloning system comprising a DNA molecule according to claim 1,functionally linked to an expression control sequence.
 8. A hostorganism transformed with a DNA molecule according to claim 1 or anexpression vector comprising it.
 9. A process for the preparation of apolypeptide encoded by a DNA sequence according to claim 1 bycultivation of a host organism and isolation of the correspondingpolypeptide from the culture.
 10. A polypeptide which is encoded by aDNA sequence according to claim
 1. 11. A polypeptide according to claim10 as medicament.
 12. A pharmaceutical composition comprising at leastone polypeptide according to claim 11 and optionally further activeingredients and/or adjuvants for the diagnosis and/or treatment ofallergies in the triggering of which group 4 allergens from the Poaceaeare involved.
 13. Use of at least one polypeptide according to claim 11for the preparation of a medicament for the diagnosis and/or treatmentof allergies in the triggering of which group 4 allergens from thePoaceae are involved and/or for the prevention of such allergies.
 14. ADNA molecule according to claim 1 as medicament.
 15. A recombinantexpression vector according to claim 7 as medicament.
 16. Apharmaceutical composition comprising at least one DNA moleculeaccording to claim 14 or at least one expression vector comprising itand optionally further active ingredients and/or adjuvants for theimmunotherapeutic DNA vaccination of patients with allergies in thetriggering of which group 4 allergens from the Poaceae are involvedand/or for the prevention of such allergies.
 17. Use of at least one DNAmolecule according to claim 14 or at least one expression vectorcomprising it for the immunotherapeutic DNA vaccination of patients withallergies in the triggering of which group 4 allergens from the Poaceaeare involved and/or for the prevention of such allergies.